Recent instrumental developments in mass spectrometry, such as matrix- assisted laser desorption and electrospray ionization, have enabled mass spectrometrists to investigate biological molecules with significantly higher Mr than previously possible. The combination of these techniques with chemical processing of large biopolymers such as proteins now enables the use of mass spectrometry to play a significant role in the realm of structural biology. We are currently working on several projects in collaboration with other groups, several of which will be described here. One is to determine the post-translational modifications of Apoliprotein AI (Apo AI) secreted by the human hepatoma cell line Hep G2 (J. Hoeg, NHLBI; M. Przybylski, W. Weinmann U. Konstanz). ApoAI is acylated only in primary hepatocyte cultures and well-differentiated hepatoma cell lines. It is hypothesized that acylation of nascent ApoAI is critical for protecting the hepatocyte and enterocyte that secrete ApoAI. By identifying the specific amino acids acylated, site-directed mutagenesis of ApoAI can be used to determine the physiological significance of acylation. The second project is the identification of proteolytic fragments of p21 implicated in cell apoptosis (Y. Yin/C.Barrett, LMC). The protein p21 is induced by p53 which is critical to cell cycle control. p21 undergoes further processing under oxidative stress to generate two smaller fragments of ca. 14 (designated p21Fc) and 6 kD (p21FN). The formation of these smaller peptides has been proposed to play an essential role in cell death. Our role in this project is to identify the p21 fragments. A third project, just being initiated is the identification of the cysteine residue(s) specifically biotinylated (M. Bernier, LCP/GRC/NIA/NIH). This one (or possibly two) cysteine residue, when biotinylated, significantly altered the insulin receptor function. We propose to biotinylate the receptor, digest, and then isolate the biotinylated residue by biotin affinity chromatography. MALDI/MS analysis of the affinity bound analytes should identify the site of activity.